Background
Malaria kills many children annually and is endemic in more than 100 countries [
1,
2]. The spread of resistant malaria parasites to anti-malarial drugs, including artemisinin-based combination therapy, is a major concern for malaria control in endemic countries [
3]. In the absence of effective vaccine, chemotherapy remains one of the strategies to control malaria. Continuous monitoring of current anti-malarial drugs is needed to detect the spread of resistant parasites strains and estimate the risk of therapeutic failure.
Three main approaches including
in vivo trials,
in vitro/ex vivo assays and molecular markers of drug resistance are currently used to monitor anti-malarial drug efficacy and drug resistance [
4].
In vivo studies are considered the gold standard for measuring the efficacy of anti-malarial drugs [
5]. They allow the measurement of clinical and parasitological efficacy of anti-malarials drugs and the detection of changes in treatment outcomes. Carrying out
in vivo efficacy trials requires time, qualified medical staff and financial resources, which can represent a challenge in resource-limited malaria-endemic countries. In addition,
in vivo trials measure therapeutic efficacy rather than drug resistance [
6,
7].
In vitro/ex vivo assays are indispensable means for measuring the intrinsic susceptibility of malaria parasites to anti-malarial drugs, and establishing baseline susceptibility of local parasite isolates to newly introduced drugs. Several techniques for
in vitro/ex vivo tests have been developed for decades. These techniques include the WHO microtest, the isotopic based tests, the enzyme-linked immunosorbent assay (ELISA) and the SYBR green-based tests [
8,
9]. However, the availability of these techniques is limited in low-income countries due to financial cost, time consumption and problems related to the management of radioactive waste. The existing
in vitro/ex vivo assays, in particular those that are non-radioactive, need to be standardized [
8]. SYBR Green I
in vitro/ex vivo assay offers a rapid, reproducible and cheap alternative to radioisotopic methods [
4,
10,
11]. This also requires specific material and expertise commonly not available in field laboratories.
Here is the report of a modified SYBR green I protocol to perform in vitro/ex vivo tests in two steps: doing parasite culture on 96-well plate containing anti-malarial drugs in standard conditions, and postponing the step of SYBR green fluorescence assay for outsourcing with specialized laboratories. Such a standardized protocol for conducting in vitro/ex vivo drug sensitivity assays for field monitoring of drug-resistant malaria would allow direct comparisons of in vitro/ex vivo results from different laboratories involved in networks of anti-malarial drugs testing. It would also allow collecting more data during field studies.
Results
Overall, three
in vitro tests were performed for each clone. The IC
50 means calculated respectively by standard and modified protocols are summarized in Table
2. No significant differences were seen in IC
50s generated for
in vitro tests between the standard and modified protocols.
Table 2
The means of CI
50
of the SYBR green based
in vitro
anti-malarial drugs assay according to protocols
3D7 | | | | | |
CQ | 31.10 ± 6.02 | 24.28-37.91 | 32.43 ± 4.31 | 26.22-35.97 | 0.77 |
DHA | 2.30 ± 0.68 | 1.53-3.06 | 1.93 ± 0.43 | 1.81-2.78 | 0.47 |
PYD | 8.82 ± 1.17 | 7.49-10.14 | 8.89 ± 1.54 | 7.07-10.56 | 0.97 |
PPQ | 26.33 ± 1.71 | 21.38-28.27 | 25.66 ± 6.82 | 15.61-34.4 | 0.88 |
W2 | | | | | |
CQ | 467.66 ± 51.18 | 409.74-525.57 | 500.66 ± 87.22 | 368.96-566.35 | 0.60 |
DHA | 1.94 ± 1.03 | 0.77-3.10 | 1.81 ± 0.99 | 0.81-3.06 | 0.81 |
PYD | 5.35 ± 1.27 | 3.90-6.79 | 4.33 ± 1.21 | 3.98-6.71 | 0.37 |
PPQ | 19.50 ± 2.91 | 16.20-22.79 | 17.20 ± 3.14 | 15.94-23.05 | 0.40 |
HB3 | | | | | |
CQ | 37.92 ± 6.66 | 30.38-45.47 | 50.28 ± 16.77 | 31.30-69.26 | 0.33 |
DHA | 2.10 ± 0.97 | 0.99-3.20 | 1.99 ± 0.41 | 1.53-2.46 | 0.87 |
PYD | 11.78 ± 2.36 | 9.10-14.46 | 12.51 ± 3.40 | 8.65-16.36 | 0.70 |
PPQ | 21.70 ± 1.76 | 19.70-23.70 | 18.51 ± 2.24 | 15.97-21.06 | 0.12 |
7G8 | | | | | |
CQ | 416.98 ± 92.57 | 311.82-521.33 | 420.55 ± 96.04 | 311.87-529.24 | 0.18 |
DHA | 0.65 ± 0.17 | 0.45-0.85 | 0.73 ± 0.12 | 0.58-0.87 | 0.56 |
PYD | 4.43 ± 1.39 | 2.84-6.01 | 5.05 ± 0.34 | 4.66-5.44 | 0.52 |
PPQ | 17.82 ± 3.57 | 13.77-21.87 | 15.98 ± 1.22 | 14.59-17.38 | 0.47 |
Ex vivo susceptibility of
P. falciparum was tested for nine samples from patients. The average culture success rate was 61.5% (ranged from 55.5-66.6%). The IC
50 were calculated respectively by standard and modified protocols and the results are summarized in Table
3. No significant differences were seen in IC
50s generated for
ex vivo samples between the standard and modified protocols.
Table 3
Ex vivo
susceptibility of
Plasmodium falciparum
isolates to chloroquine, dihydroartemisinin, pyronaridine and piperaquine (standard and modified protocols)
CQ | 66.6% (6/9) | 484.08 [176-791] | 730.66 [595-995] | 0.35 |
DHA | 66.6% (6/9) | 1.67 [1.21-2.12] | 1.44 [0.88-2] | 0.55 |
PYD | 55.5% (5/9) | 14.23 [2.83-25.63] | 16.85 [1.69-32] | 0.79 |
PPQ | 55.5% (5/9) | 78.22 [36.56-119.87] | 74 [24.61-123.38] | 0.90 |
Discussion
Among the different
in vitro/ex vivo assay methods, the use of fluorescent labeling of DNA with SYBR Green offers some advantages compared to methods that use tritiated hypoxanthine or ELISA [
4,
10-
12]. However, the fluorescence determination requires material not commonly available in field laboratories. Postponing the step of fluorescence determination allows the use of SYBR Green I protocol in field laboratories with limited resources. It also offers opportunity (i) to do simultaneously
ex vivo and
in vivo tests during field studies, (ii) to standardize protocols for data collection during multicenter clinical studies, (iii) to store data for future use, (iv) and to share original data with collaborators. The SYBR Green I modified protocol described in this study enables to dry and store the plates, which will be analysed later by well-equipped laboratories. Networks devoted to anti-malarial drugs resistance assay [
13-
17], may use this modified protocol for multicentre clinical studies, that could result in reduction of instrumental and systematic bias and facilitate the compilation, analysis and interpretation of data from different study sites. The risks of environment pollution are scarce and the technique does not require specific protection of manipulator since the SYBR Green is not radioactive [
8] while its toxic for DNA, and will be added to the culture in the laboratories where the fluorescence will be read.
The plates were dried at 50°C to minimize the risk of contamination of the culture with bacteria or fungi (higher temperatures could eliminate the risk of contamination, but they could denature the DNA of the parasite). The modified protocol was first tested with laboratory clones
in vitro, then with field fresh isolates
ex vivo. Samples were collected from nine imported malaria cases (six from Cameroon and three from the Democratic Republic of Congo). Four patients have declared the use of chemoprophylaxis against malaria (with chloroquine or chloroquine-proguanil) prior to admission. The average of culture success rate (interpretable tests) of
ex vivo assays was 61.5%, slightly lower than this described by Tinto
el al. (85%) [
18], probably due to the small sample size of the study reported here.
There was no significant difference between the means of IC50 determined by the standard and modified protocols. The values of IC50 determined in vitro by the modified protocol were broadly similar to those calculated using the standard protocol (p > 0.10). The duration of storage did not affect the IC50 value (IC50 of CQ against 7G8 ranged from 676 nM in standard protocol to 502 nM after 24 hours and 9 days of storage of dried plates in modified protocol). A slight but not significant decrease was observed in IC50 values of dried plates. These findings suggest that the dried plates can be stored at room conditions and be sent without special transportation requirements since biological material is stable and non-infectious.
The means of IC
50 of chloroquine against sensitive clones 3D7 (32.43 nM, 95% CI: 26.22-35.97) and HB3 (50.28 nM, 95% CI: 31.3-69.2) determined
in vitro by the modified SYBR Green I protocol are broadly similar to those already described
in vitro by Garbi et al. in Senegal, Mali, Cameroon and Côte d’Ivoire [
19] and Issaka et al. in Niger [
20]. For
ex vivo assays, our IC
50 values of CQ (484.08 and 730.66 nM) are also comparable to the maximum values described by Tinto et al. (using the semiautomated microdilution technique) in Burkina Faso (8.3-595.9 nM) [
18]. The lowers values (8.3 nM) obtained by Tinto et al. could be the fact that resistant and sensitive strains are circulating simultaneously in populations, and the large sample size included in this study.
The means of IC
50 of the chloroquine were high in
ex vivo assays (484.08 and 730.66 nM respectively in standard and modified protocols). Malaria cases were imported from chloroquine-resistance areas. High resistance rates of
P. falciparum to chloroquine have been described in Cameroon since 2000 [
21], even though recent studies have shown decreases in resistance due to changes in malaria treatment policies in these areas [
19].
Conclusion
Drying and storing the plates did not affect the means of IC50 values in a modified SYBR Green I protocol to assess anti-malarial drugs. The modified protocol allows performing in vitro/ex vivo tests in two steps. By postponing the step that requires SYBR Green methodology, the tests could be done in field conditions and the dried plates could be transported in ambient conditions. Field staff involved in multicentre studies may use this protocol to standardize the collect and share of data. It offers the possibility to do simultaneously clinical in vivo and ex vivo/in vitro assays in field. More in vitro/ex vivo studies with large sample size are necessary for the validation of this protocol.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SP, FG, KT designed the study and experiments, KT, AL, GB, MC, FS, GCB performed data collection; SP, OKD, FG and KT performed data analysis and wrote the manuscript. All the authors have read and approved the content of this manuscript.